Vitamin D regulates bone metabolism and may exert pleiotropic effects on other physiological systems with potentially important implications for health outcomes. Funding from our soon-to-expire R01 (DL094486-02) enabled our team to investigate a novel hypothesis about bioavailable vitamin D (BavD), the fraction not bound to vitamin D binding protein (DBP) that is free to enter cells and exert biologic effects (Powe et al. 2013, New England Journal of Medicine). Using blood samples obtained from a large cohort of black and white subjects (n=2,085) from the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS, National Institutes of Aging) study, we found that black Americans, despite having lower total 25D than whites, have similar BavD when taking into account: (1) genetic variations in DBP (Gc1F, Gc1S), (2) the abundance of DBP variants in the circulation, and (3) DBP-affinity binding constants. Our conclusions relied on calculated BavD values based on measured DBP concentrations and published affinity constants and were therefore applicable only to homozygous individuals. Despite this limitation, our findings advanced the current understanding of vitamin D biology, possibly explaining the paradoxical relationship between vitamin D and bone density in blacks vs whites and the impact of race on measures of vitamin D status. Our findings also raise important questions about how vitamin D deficiency is defined, and have been independently described as having profound implications for interpretation of vitamin D levels. An extension of funding will allow us to utilize a novel assay that we developed to directly measure BavD, and strengthen our conclusions. We will eliminate a key limitation of our study, i.e., reliance on calculated BavD. In doing so, we have the potentia to directly impact recommendations for vitamin D intake set forth by the Institute of Medicine (IOM) and other health policy- makers. Moreover, we will improve upon existing methods for measuring circulating total 25-hydroxyvitamin D (25D). Unlike total 25D levels which are the current standard clinical test, our new approach takes the bioavailable fraction of 25D into account, and is independent of race or genotype. We plan to directly measure BavD in: (1) the racially-diverse HANDLS cohort to confirm whether BavD is the universal marker of choice in all racial groups to determine true vitamin D status; (2) women in the menopausal transition (SWAN cohort), to evaluate whether BavD reflects bone health better than total 25D; (3) chronic kidney disease patients with a wide range of parathyroid hormone levels to evaluate thresholds that define normal vs abnormal cutoff levels for BavD. We plan to complement these clinical studies by creating 'humanized' animal models to prove that genetic polymorphisms resulting in DBP variant phenotypes (Gc1F, Gc1S) produce differences in total 25D and BavD depending on the vitamin D content of the diet. Our team (clinical researchers/epidemiologist, clinical chemist and basic scientist) have complementary skills and are uniquely qualified to carry out the specific aims proposed in this grant renewal application.